High Resolution
Melting (HRM) is
a novel,
homogeneous, close-tube, post-PCR
method, enabling genomic researchers to analyze genetic variations
(SNPs, mutations, methylations) in PCR amplicons. It goes beyond the
power of classical melting curve analysis by allowing to study the
thermal denaturation of a double-stranded DNA in much more detail and
with much higher information yield than ever before.
HRM
characterizes nucleic acid samples based on their disassociation
(melting) behavior. Samples can be discriminated according to their
sequence, length, GC content or strand complementarity. Even single
base changes such as SNPs (single nucleotide polymorphisms) can be
readily identified.

The most important High Resolution
Melting application is gene scanning - the search for the presence of
unknown variations in PCR amplicons prior to or as an alternative to
sequencing. Mutations in PCR products are detectable by High Resolution
Melting because they change the shape of DNA melting curves. A
combination of new-generation DNA dyes, high-end instrumentation
and
sophisticated analysis software allows to detect these changes and to
derive information about the underlying sequence constellation.

HRM
ApplicationsThe
introduction of HRM has renewed interest in the utility of DNA melting
for a wide range of uses, including:

Mutation
discovery (gene scanning)

Screening
for loss of heterozygosity

DNA
fingerprinting

SNP
genotyping

Characterization
of haplotype blocks

DNA
methylation analysis

DNA
mapping

Species
identification

Somatic
acquired mutation ratios

HLA
compatibility typing

Association
(case/control) studies

Allelic
prevalence in a population

Identification
of candidate predisposition genes

With HRM, these
and other applications are done using low-cost generic dyes where
previously custom labeled probes such as TaqMan® or fluorescence
resonance energy transfer (FRET) probes were required. HRM is thus a
simpler and much more cost-effective way to characterize samples.

In molecular biology High Resolution Melt or HRM
analysis as it will
be referred to herein is a hugely powerful technique for the detection
of mutations, polymorphisms and epigenetic differences in double
stranded DNA samples. It has advantages over other genotyping
technologies. Namely:

It is massively cost effective vs. other genotyping
technologies
such as sequencing and Taqman SNP typing. This makes it ideal for large
scale genotyping projects.

It is fast and powerful thus able to accurately
genotype huge numbers of samples in rapid time.

It is simple. With a good quality HRM assay powerful
genotyping can
be performed by non-geneticists in any laboratory with access to an HRM
capable real-time PCR machine.

For several years, various researchers and instrument makers have
independently investigated the utility of high-resolution DNA
dissociation analysis. For example, the team at Idaho Technology has
done an admirable job of vigorously promoting their research through
traditional journal publications. Conversely, Corbett Life Science does
not pursue publication, but instead relies on the publications of
customers to promote the technology. Regardless, both companies have
independently advanced the field of high resolution dissociation
analysis and successfully introduced what has now become known as high
resolution melt (HRM) analysis.

Idaho
Technology was first to market with an instrument made
specifically to do dissociation analysis; the HR-1. The HR-1 was a
showpiece for the technology with the singular aim of producing the
most detailed melt curve possible. As such, it opened the eyes of many
to the potential of HRM and remains the performance benchmark for the
acquisition of an individual melt curve. However the HR-1 is not
capable of thermal cycling and can only analyze a single sample from
within a glass capillary per run making data analysis time consuming. http://www.idahotech.com/HR-1/index.html

Multi-well instruments with greater practical utility were introduced
to the market very soon after the HR-1. The first multi-well HRM
instruments were the Rotor-Gene
6000 (Corbett Life Science) and the LightScanner
(Idaho Technology)(PDF). These
two instruments were introduced
at about the same time but employed fundamentally different technical
innovations to achieve HRM. The LightScanner uses a modified
block-based design available in 96-well or 384-well versions. Despite
advanced engineering, it still suffers from measurable sample-to-sample
thermal and optical variation and is unable to match the performance
benchmark set by the original HR-1 instrument. Like the HR-1, the
LightScanner is not capable of thermal cycling.

The Rotor-Gene 6000 was the first of
the multi-well instruments capable
of both thermal cycling and HRM. This dual capability enables samples
to be fully processed in the one instrument (i.e. pre-amplification and
HRM done consecutively in the one run). A major advantage of this is
that amplification plots can be used to help interpret HRM results
since aberrant amplification plots (i.e. those that amplified
differently to what was expected) also produce aberrant HRM data. In
this way compromised samples can be easily identified and removed from downstream HRM
analysis. The main advantage of the Rotor-Gene for HRM
stems from its rotary design, in which samples spin under centrifugal
force past a common optical detector. This is seemingly ideal for HRM
as thermal or optical variation between samples is insignificant. The
result is that the Rotor-Gene HRM performance closely matches the HR-1
benchmark with the compromise that samples are not arranged in a
conventional array format (as they are in block-based instruments) but
are instead arranged around the perimeter of a spinning rotor.

The more recently introduced LightCycler
480 (Roche Molecular Systems)
is capable of HRM and thermal cycling. The LightCycler 480 is
a block-based instrument design and it
has better thermal uniformity than other block-based instruments, it
nevertheless does exhibit measurable thermal and optical non-uniformity.

Other instrument providers are now rushing to introduce HRM capability
and some are planning to release software upgrades to support HRM
analysis. The danger here is that instruments not specifically
engineered for HRM will deviate so much from the HR-1 performance
benchmark that careful investigation will need be done before accepting
those instruments as HRM capable.

Example HRM data for
each of the multi-well HRM systems discussed here
is shown in the figures (A-E) below.

For comparison
purposes, similar data for two standard
real-time PCR instruments (i.e. not engineered for HRM) is also shown.
All data has been enlarged without modification directly from (Herrmann et al 2007)
Normalized melting curves of a 110 bp beta-globin amplicon
(triplicate HRM data) containing single and double SNPs are shown.

by “Shape”
,
i.e. using detail in the shape of the melt curve itself and by “Shift”;
i.e.
the thermal offset of a curve from other curves.

Before HRM curves are plotted, the raw data is first normalized. Melt
curves are normally plotted with fluorescence on the Y axis and
temperature on the X axis. This is similar to real-time PCR
amplification plots but with the substitution of temperature for cycle
number. As with real-time PCR plots, the fluorescence axis of HRM plots
is normalized onto a 0 to 100% scale.

An emerging trend is to also apply normalization to the temperature (X)
axis. This has the desired effect of compensating for well-to-well
temperature measurement variations between samples. Known as
“temperature shifting”, it was introduced by Idaho Technology and is
now also supported by the Roche LightCycler 480. Unfortunately,
temperature shifting normalization removes any potential discriminatory
power provided by the temperature data.

For some applications, temperature shifting normalization may be a
useful solution but for many routine applications it is actually
detrimental. A good example of this is the discrimination of homozygous
SNPs. On the one hand, heterozygous samples are often more easily
discriminated after temperature shifting normalization (because their
curves have a complex shape), but the discrimination of homozygous
samples is usually made more difficult because they often have a simple
and identical curve shape (Figure 1).
While homozygous SNP samples have an identical curve shape, they can
usually be discriminated by HRM analysis by observing a change in their
respective Tm’s. This characteristic means the melt plots of different
homozygotes will be offset one from another thereby allowing them to be
readily discriminated (so long as temperature shifting normalization is
not applied and the HRM temperature data is precise enough). Currently,
the only instrument system that does not use temperature shifting
normalization and can reliably discriminate homozygous SNPs is the
Rotor-Gene (Corbett Life Science). The Rotor-Gene can discriminate
homozygotes because well-to-well thermal variation is so low on that
instrument that the collected temperature data is sufficiently precise (Figure 2).

Triplicate
HRM data
was captured on a Rotor-Gene for SNP genotyping (Herrmann et al 2007)
Normalized melting curves are of a 110 bp beta-globin amplicon. Each
category of SNP genotype can be readily discriminated prior to thermal
shifting normalization. However, when curves are thermal shifted the
homozygous genotypes overlay precisely and can no longer be
discriminated.

High-resolution DNA
Melting Analysis

When it comes to
genotyping and mutation scanning, high-resolution DNA melting is
emerging as the technique of choice because it is inexpensive simple,
accurate and rapid. Development of this method of DNA analysis
has been underway since its introduction in 2002 by a team of
researchers from our Pathology Department led by Dr. Carl Wittwer and
Dr. Karl Voelkerding at the University of Utah coupled with
collaborative efforts from Idaho Technology. High-resolution melting
required new instrumentation. The first high-resolution
instrument developed, named the HR-1, remains the most accurate with
the fastest analysis speed, while the LightScanner has the highest
throughput. In addition to the special instrumentation, high-resolution
melting uses special saturation dyes that fluoresce only in the
presence of double stranded DNA. These dyes are included in the
PCR amplification process. When the sample is heated to high
temperatures, the DNA denatures and the fluorescent color fades away as
the double stranded DNA separates, generating a melting curve. Because
different genetic sequences melt at slightly different rates, they can
be viewed, compared, and detected using these curves. Even a
single base change will cause differences in the melting curve.
The process can be used for specific genotyping, comparing sequence
identity between two DNA samples, and scanning for any sequence variant
between two primers. High-resolution DNA
melting is becoming more popular as its
accuracy and simplicity is recognized. High-res DNA melting makes
it
possible to quickly and accurately determine whether DNA sequences
match, providing an interesting option for transplantation matching and
forensics. Genotyping via
high-resolution melting is more streamlined and
less expensive than methods that use complex probes.

No processing is
required, and when combined with the speed of rapid-cylce PCR, has
interesting potential for personal DNA diagnostics.For
example, the
amount of medication a person needs is often dependent on sequence
variants in genes that can be determined through high-resolution DNA
melting. Hi-res melting can also be used to scan large genes for
variation, in
many cases greatly reducing or eliminating the need for sequencing.
Although high-resolution DNA melting is relatively new, it is expanding
and being improved upon by our talented team of scientists in Pathology
and we are excited to be at the forefront of such innovative and
important technology.

High Resolution Melting (HRM) analysis is an
alternative to dHPLC
sequencing screening of new gene variants. The HRM Software is now
available on the Applied Biosystems 7500 Fast System and on the 7900HT
Fast Real-Time PCR System. The 7500 Fast Real-Time PCR System delivers
precise results with fast thermal cycling in a standard 96-well format.
Achieve high-throughput HRM analysis with the 384-well 7900HT, the gold
standard high throughput system. The AB HRM application does not
require temperature shifting, which results in a greater likelihood of
identifying new homozygous mutations than methods that require
temperature shifting.

The
ability to easily identify new variants is key for successful HRM
applications. By eliminating the temperature shift step, the Applied
Biosystems HRM solution (Fig 1) was able to clearly distinguish
homozygous variant samples from homozygous wildtype samples in 97.5% of
the population, whereas the other HRM system from Competitor R (Fig 2)
was only able to distinguish them in 10% of the population. All
genotypes were auto-called by the respective software packages and were
not altered by the operator. Class 1 SNP (A/G), multiple technical
replicates of nine DNA samples representing three genotypes: homozygous
wildtype (G/G), homozygous mutant (A/A) and heterozygous (A/G).

a software algorithm that analyzes the shape of the
melting curves and groups those that are similar.

In a Gene Scanning experiment, sample
DNA is
first amplified via
real-time PCR in the presence of a proprietary saturating DNA dye. A
melting curve is then performed using high data acquisition
rates, and data are finally analyzed using a Gene Scanning Software, by
three basic steps:

Normalization:the pre-melt (initial
fluorescence) and post-melt
(final fluorescence)
signals of all samples are set to uniform, relative values from 100% to
0%

Temperature
shifting: the
temperature axis of the normalized melting curves is shifted to the
point where the entire double-stranded DNA is completely denatured.
Samples with heterozygous SNPs can then be easily be distinguished from
the wild type by the different shapes of their melting curves.

Difference Plot:
the differences in melting curve shape are further analyzed by
subtracting the curves from a reference curve. This helps cluster
samples automatically into groups that have similar melting curves
(e.g., those who are heterozygote as opposed to homozygotes).

Roche Applied
Science´s LightCycler® family of real-time PCR systems offer
fast, accurate and versatile platforms for genetic
variation research. The new plate-based LightCycler® 480 System
provides the temperature
homogeneity and optical characteristics required for high-performance
melting-curve analysis (MCA). On
the level of data acquisition and available detection channels, this
new instrument opens the way to more
advanced applications in the emerging field of gene scanning where
amplicons can be screened for
unknown sequence variations with low efforts in time and cost.The LightCycler® 480
real-time PCR system: a versatile platform for genetic variation
research
Real-time PCR is a well established technique for studying genetic
variation using various probe-based methods for genotyping as well as
high-resolution analysis of whole amplicons melted in the presence of
saturating DNA dyes. The latter, relatively new, method allows
screening for unknown mutations or DNA modifications. The
LightCycler® 480 real-time PCR system is a multiwell plate–based
instrument that provides integrated applications for detecting and
characterizing genetic variation using all these methodological
approaches.

Transfering
PCRs
to HRM-assays on the LightCycler 480 System- Examples
for BRCA1
High-resolution melting curve analysis (hrMCA) is an attractive
technique to scan for unknown mutations in genes. To evaluate how easy
or difficult it is to design hrMCA assays using the LightCycler®
480 Instrument, we selected 3 different fragments in exon 11 of the
BRCA1 gene, designed an MCA assay, and tested its sensitivity to detect
known variants.

Recently,
HRM was the subject of a detailed and independent Technology Assessment
report from the National Genetics Reference Laboratory (Wessex, UK). A
wide range of sample types were tested, including examples of
challenging G to C and A to T single base substitutions. The full
report is now available for download =>

Classifying and understanding genetic variation
between populations and individuals is an important aim in the field of
genomics. Many common diseases (diabetes, cancer, osteoporosis, etc.)
and clinically relevant phenotypic traits are elicited from the complex
interaction between a subset of multiple gene products and
environmental factors. High resolution melt (HRM) analysis is the
quantitative analysis of the melt curve of a DNA fragment following
amplification by PCR and can be considered the next-generation
application of amplicon melting analysis. It is a low-cost, readily
accessible technique that merely requires a real-time PCR detection
system with excellent thermal stability and sensitivity and
HRM-dedicated software. However, careful sample preparation and
planning of experimental and assay design are crucial for robust and
reproducible results. The following guidelines assist in the
development of such assays.